Karyopherin α2-dependent import of E2F1 and TFDP1 maintains protumorigenic stathmin expression in liver cancer

Abstract

Background: Members of the karyopherin superfamily serve as nuclear transport receptors/adaptor proteins and provide exchange of macromolecules between the nucleo- and cytoplasm. Emerging evidence suggests a subset of karyopherins to be dysregulated in hepatocarcinogenesis including karyopherin-α2 (KPNA2). However, the functional and regulatory role of KPNA2 in liver cancer remains incompletely understood.

Methods: Quantitative proteomics (LC-MS/MS, ~ 1750 proteins in total) was used to study changes in global protein abundance upon siRNA-mediated KPNA2 knockdown in HCC cells. Functional and mechanistic analyses included colony formation and 2D migration assays, co-immunoprecipitation (CoIP), chromatin immunoprecipitation (ChIP), qRT-PCR, immmunblotting, and subcellular fractionation. In vitro results were correlated with data derived from a murine HCC model and HCC patient samples (3 cohorts, n > 600 in total).

Results: The proteomic approach revealed the pro-tumorigenic, microtubule (MT) interacting protein stathmin (STMN1) among the most downregulated proteins upon KPNA2 depletion in HCC cells. We further observed that KPNA2 knockdown leads to reduced tumor cell migration and colony formation of HCC cells, which could be phenocopied by direct knockdown of stathmin. As the underlying regulatory mechanism, we uncovered E2F1 and TFDP1 as transport substrates of KPNA2 being retained in the cytoplasm upon KPNA2 ablation, thereby resulting in reduced STMN1 expression. Finally, murine and human HCC data indicate significant correlations of STMN1 expression with E2F1/TFPD1 and with KPNA2 expression and their association with poor prognosis in HCC patients.

Conclusion: Our data suggest that KPNA2 regulates STMN1 by import of E2F1/TFDP1 and thereby provide a novel link between nuclear transport and MT-interacting proteins in HCC with functional and prognostic significance.

Keywords: E2F1; HCC; Karyopherin; Nuclear transport; Stathmin; TFDP1.

Fig. 1

Stathmin is downregulated upon KPNA2 depletion. a Workflow of LC-MS/MS analysis. HLE cells were harvested 72 h after control (Ctrl.) or KPNA2 siRNA treatment (n = 3). b Volcano-Plot illustrates the resulting log₂ fold-changes (KPNA2/Ctrl. siRNA) and corresponding log₁₀ p-values of 1759 proteins being quantified by the LC-MS/MS analysis. Horizontal dotted line p = 0.01; vertical dotted lines log₂ fold-change 0.8 or − 0.8; green dot: KPNA2; red dot: stathmin. c HLE and HLF cells were siRNA-treated and harvested as described in (a). Lysates were immunoblotted using the indicated antibodies. d HLE and HLF cells were treated as described in (a) and STMN1 expression was analyzed by qRT-PCR. (HLE: n = 5, p < 0.01 (**); HLF: n = 4, p < 0.05(*))

Fig. 2

KPNA2 depletion reduces the clonogenic capacity and cell migration of HCC cells being phenocopied by stathmin knockdown. a, b HLE cells were treated either with ctrl. or KPNA2 siRNAs and colony formation was analyzed 14 days after treatment by crystal violet staining (n = 4; p < 0.05 (*)). c, d HLE cells were treated either with ctrl. or stathmin siRNAs and colony formation assays were performed as described in (a) (n = 4; p < 0.05 (*)). e, f HLE cells were treated as described in (a) and two-dimensional scratch assays were started 48 h later. Gap closure was analyzed 18 h after scratching (n = 4; p < 0.05 (*)). g, h HLE cells were treated as described in (c) and scratch assays were performed as described in (e) (n = 4; p < 0.05 (*))

Fig. 3

KPNA2 regulates STMN1 by import of the transcription factors E2F1 and TFDP1. a HLE cells were treated with ctrl. or KPNA2 siRNAs and nuclear-cytoplasmic fractionation was performed after 72 h. Samples were immunoblotted using the indicated antibodies. b HLE cells were co-transfected with HA-tagged KPNA2 and Flag-tagged E2F1 or TFDP1. KPNA2 immunoprecipitation was performed and samples were immunoblotted using the indicated antibodies. c, d HLE cells were treated with ctrl. siRNA or siRNAs directed against E2F1 or TFDP1 and STMN1 expression was analyzed by immunoblotting (upper panel) or qRT-PCR (lower panel, n = 4; p < 0.05 (*)). e HLE cells were treated with siRNAs directed against E2F1 and TFDP1 and STMN1 expression was analyzed by immunoblotting (upper panel) or qRT-PCR (lower panel, n = 4; p < 0.05 (*)). f Illustration of the predicted E2F1 binding sites (BS) within the promoter region of STMN1. A non-coding region downstream of the promoter region served as negative control. g E2F1 was immunoprecipitated in HLE cells, ChIP assay was performed and precipitated DNA of the predicted STMN1 bindings sites, the positive control binding site (CDC2) and a control region (neg ctrl) was quantified using qRT-PCR. The bar diagram depicts one representative experiment. h TFDP1 was immunoprecipitated in HLE cells and ChIP assay was performed as described in (g). The bar diagram depicts one representative experiment

Fig. 4

KPNA2 and stathmin/STMN1 expression are correlated in human HCC. a Representative micrographs show human HCC samples either H&E stained (upper row) or immunostained with KPNA2 (middle row) or stathmin (lower row). Scale bar: 100 μm. b Boxplots illustrate increasing immunohistochemical (IHC) scores of KPNA2 (upper panel) or stathmin (lower panel) with tumor dedifferentiation (G1 = well differentiated, G2 = moderately differentiated, G3–4 = poorly differentiated). c Spearman correlation between KPNA2 and STMN1 mRNA expression in a large HCC cohort (Roessler cohort). STMN1 is correlated to E2F1 and TFDP1 in human HCC. d, e Spearman correlation between STMN1 and E2F1 (d) or TFDP1 (e) expression in human HCC samples (Roessler cohort). High expression of KPNA2 and STMN1 correlates with poor prognosis in HCC patients. Overall survival of HCC patients showing low and high mRNA expression of KPNA2 (f) and STMN1 (g) or both (h) (cut-off: median, Roessler cohort)

Fig. 5

KPNA2 drives protumorigenic STMN1 expression by nuclear import of the transcription factors E2F1 and TFDP1. E2F1 and TFDP1 form a heterotrimeric complex with KPNA2 and importin β1 (KPNB) which translocates into the nucleus through the nuclear pore complex (NPC). Upon dissociation of the complex E2F1 and TFDP1 bind to the STMN1 promoter and drive STMN1 expression (red wavy lines = STMN1 mRNA). Compared to a normal, healthy liver KPNA2 is overexpressed in HCC, resulting in accelerated E2F1/TFDP1-mediated STMN1 transcription